Cell culture
The mouse neuroblastoma x spinal cord hybrid cell line (NSC-34 cells [52]) were routinely cultured in DMEM/F12 supplemented with 10 % (v/v) FBS and 2 mM GlutaMAX. Cells were maintained in an incubator at 37 °C under a humidified atmosphere containing 5 % (v/v) CO2. Human fibroblasts were sourced from non-ALS individuals (female aged 62 and male aged 59 at the time of collection) and reprogrammed into induced pluripotent stem cells using mRNA (Miltenyi). Pluripotency was confirmed by PluriTest and differentiation of the cells [53]. Karyotyping was carried out in iPSCs, to ensure chromosomal abnormalities were not introduced during reprogramming and culture. Immunocytochemistry confirmed the expression of the pluripotency marker Oct4 (Additional file 9A).
Differentiation of pluripotent stem cells into motor neurons was carried out as in [54] and one clone from each line was used in experiments. Motor neurons (150,000 cells) were plated onto laminin (20 μg/mL) and fibronectin (10 μg/mL) coated 13 mm coverslips. The timeline summarizes the differentiation stages and the growth factor conditions used during differentiation (Additional file 9B). The morphological changes of each cell line were examined at each stage of the differentiation process (Additional file 9B).
Confirmation of motor neuron phenotype was carried out, including expression analysis by quantitative reverse transcription PCR and immunocytochemistry. The differentiated neurons expressed the motor neuron specific markers SMI32 and islet 1 (Additional file 9C). Immunocytochemistry identified the presence of extended dendrites ~100 μm in length. Quantitative reverse transcription PCR analysis identified the expression of the motor neuron specific gene MNX1 (that encodes the transcription factor homeobox 9, HB9) [55]. MNX1 was specifically expressed in motor neurons and MNX1 was silent in pluripotent stem cells. The cholinergic specific marker acetylcholine esterase (ACHE that encodes the enzyme responsible for the degradation of the neurotransmitter acetylcholine) was specifically expressed in cholinergic motor neurons. The expression levels for both MNX1 and ACHE, were normalized to the housekeeper gene GAPDH (Additional file 8D).
Application of aggregates to cells
Wt and G93A SOD1 were expressed and purified from E.coli as previously outlined [50, 56]. SOD1 aggregation was performed in vitro as previously described [50]. Briefly, solutions of purified wt or G93A mutant SOD1 protein (1 mg/mL) in PBS were co-incubated with 20 mM dithiothreitol (DTT) and 5 mM ethylenediaminetetraacetic acid (EDTA) for 72 h at 37 °C with shaking; aggregated SOD1 was washed several times to remove DTT and EDTA. NSC-34 cells were cultured in 12 well plates and were transfected with wt or mutant SOD1-GFP using lipofectamine 2000 (following the manufacturer’s instructions). Lipofectamine was removed after 5 h and replaced with 10 % FCS in DMEM. After 24 h the aggregates, or soluble (non-aggregated) wtSOD1 as a control, were added in fresh media to transfected or naïve NSC-34 cells. Cells were incubated for a further 48 h and then imaged. In other experiments, aggregates were added to untransfected NSC-34 cells and incubated for various time periods in the presence or absence of pathway inhibitors before fixation and detection of aggregates (see online methods for details). In some experiments, NSC34 cells were incubated with 20 μg/mL of human wt and mutant SOD1 aggregates for 1 h at 37 °C. Post incubation, cells were washed three times in PBS and incubated with trypsin (0.25 %, Invitrogen) for 5 min to remove surface-bound aggregates. The resulting detached cells were centrifuged at 1100 × g for 5 min, re-plated in media, and allowed to recover for 6 h at 37 °C before fixation for immunocytochemistry.
Aggregation and biotinylation of wt and G93A SOD1 aggregates
SOD1 aggregation was performed in vitro as previously described 50. Aggregated SOD1 was labelled with biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt in DMSO for 2 h at RT. The unconjugated biotin was then separated by centrifugation (21 000 x g for 30 min) and washed three times with PBS. The purified aggregates were then resuspended in PBS (pH 7.4). A bicinchoninic acid protein assay was performed to determine the amount of protein in solution. Aggregated forms of other proteins were obtained by incubation under conditions previously described, Httex146Q [57] , TDP-43 [58], α-synuclein [59], and α-lactalbumin [38].
Cell surface binding and internalization of aggregated SOD1
NSC-34 cells were initially incubated with 20 μg/mL of aggregated SOD1 for 30 min at 4 °C. Cells were then fixed with 4 % (w/v) paraformaldehyde (PFA) in PBS (pH 7.4) before immunodetection. In separate experiments, cells were incubated with 20 μg/mL of aggregated wtSOD1 for 60 min at 4 °C, fixed with 4 % (w/v) PFA in PBS (pH 7.4) and permeabilized, or not, with Triton x-100. SOD1 was then detected using anti-SOD1 antibodies. Cells were imaged using a Leica TCS SPII laser scanning confocal microscope (Heidelberg, Germany). In addition, quantitative analysis of SOD1 internalisation into NSC-34 cells was performed using flow cytometry using a BD LSRII (California, USA). Cells were incubated with 20 μg/mL of aggregated SOD1 for 60 min at 4 °C. Cells incubated with PBS (pH 7.4) alone acted as the control. Similar experiments were performed in the presence of the dye, Lysotracker Red as per the manufacturer’s instructions. In addition, some cells were treated with trypsin and washed extensively before being lysed after incubation with aggregated wtSOD1 for 60 min. The cell lysates were analysed by Western blotting.
Internalisation of aggregated SOD1 was measured in the presence or absence of a range of compounds that inhibit various internalisation mechanisms. NSC34 cells were pre-treated with various endocytic inhibitors including 100 μM 5-N-ethyl-N-isopropyl-amiloride (EIPA), 5 μM chlorpromazine hydrochloride (CPZ), 10 μM genistein (Gen) or 3 μM rottlerin (Rot) diluted in 1 % BSA/PBS for 30 min at 37 °C, followed by 20 μg/mL aggregated wtSOD1 for 30 min at 37 °C. Cells were then fixed and permeabilized before detection of biotinylated aggregates with SA- Alexa 488. Cells were washed once with PBS medium and analysed using a LSR II flow cytometer (BD Biosciences, San Diego, CA) (excitation 488 nm, emission collected with 515 ± 20 band-pass filters). The mean fluorescence intensity (MFI) of relative SOD1 uptake was determined using FlowJo software (Tree Star, Ashland, OR). For confocal microscopy, cells remained on glass coverslips and incubated in wells as outlined for flow cytometry. Sytox Red (5 nM) was used as a counter stain. Results are the average of at least five independent experiments.
Field emission scanning electron microscopy (FESEM)
NSC-34 cells in phenol-red serum free culture medium were plated into 12-well plates with 19 mm glass coverslips (7x104 cells/ ml/well) and starved of serum for 24 h, and treated with 20 μg/mL soluble or aggregated proteins in PBS or PBS containing 200 nM PMA for 2 h at 37 °C. Post incubation, cells were washed three times in PBS then fixed in 2.5 % glutaraldehyde/ 4 % PFA in 0.1 M phosphate buffer (pH 7.4) for 3 h at 4 °C. The cells were then washed three times in phosphate buffer and postfixed in 2 % OsO4/ water at RT for 1 h. After washing with water, the cells were dehydrated using a gradient of ethanol at 30, 50, 70, 80, 90 and 100 % (30 min per incubation) at RT. The cells were then critical point dried for 2 h using a LEICA CPD030 (Vienna, Austria) and coated with graphite-gold in a sputter coater. The samples were analysed with a JEOL 6490LV SEM (Tokyo, Japan) operated at 10 kV at a 10 mm working distance and a spot size setting of 35.
Rac1 activation assays
NSC-34 cells were treated with 20 μg/mL of soluble and aggregated G93A SOD1 for 30 min at 37 °C. The cells were washed twice with cold PBS and harvested by treatment with 0.05 % trypsin for 10 min at 37 °C. Rac1 activation was measured using a G-LISA activation kit (Kit #BK128 Cytoskeleton, Inc. (Denver, USA) as per the manufacturer’s recommendations.
Transmission electron microscopy
Negative staining was performed using substrate carbon-coated nickel grids (Proscitech Kirwan, Australia). Protein was loaded onto the grid and washed three times with milli-Q water. Subsequently, 2 % (w/v) uranyl acetate (ProsciTech Kirwan, Australia) in 0.22 μm sterile filtered milli Q water was added for 2 min to stain the proteins negatively. The grids were analysed using a JEOL 2011 TEM (Tokyo, Japan) operated at 200 kV and Images taken using a Gatan Orius digital camera (California, USA).
Pharmacological inhibitors and antibodies
Pharmacological inhibitors were prepared in either DMSO or 20 % acetonitrile/water according to the manufacturer’s recommendations and used at the indicated concentrations. EIPA, CPZ, Gen, and Rot were purchased from Sigma Aldrich. The Rac1 inhibitor W56 was purchased from Tocris Bioscience.
Specific antibodies including mouse anti-beta actin (ab8226), rabbit anti-EEA1 antibody (ab2900), Anti-LAMP1 [H4A3], anti-beta actin antibody [AC-15], anti-beta tubulin antibody, anti-neuron specific beta III tubulin were purchased from Abcam. Alexa Fluor 488 goat anti-mouse, Alexa Fluor 488 goat anti-rabbit, streptavidin Alexa Fluor 633 conjugate, streptavidin Alexa Fluor 488 conjugate, Alexa Fluor 488 donkey anti-sheep, Alexa Fluor 488 donkey anti-rabbit, SYTOX Red dead cell stain, FM® 1-43FX fixable analogue of FM® 1–43 membrane stain were purchased from Invitrogen Life Technologies. Donkey anti-sheep/goat IgG HRP conjugate and goat anti-mouse IgM + IgG + IgA (H + L) HRP conjugates were purchased from Millipore.
Sheep anti-SOD1 was purchased from Thermo Fisher Scientific. Mouse monoclonal anti-human TARDBP antibody (clone k1B8) was purchased from Abnova. Anti-BiP/GRP78 was purchased from BD Transduction Laboratories. FITC-conjugated sheep anti-mouse was purchased from Silenus. RedDot 2 was obtained from Biotium. Goat Anti-Rabbit IgG (H + L)-HRP Conjugate was obtained from Bio-Rad.
Selective permeability of cells
NSC-34 cells were incubated with 20 μg/mL of biotinylated aggregated wt and G93A SOD1 in PBS for either 60 or 120 min at 37 °C. Post incubation, cells were fixed and permeabilized with either 10 μM digitonin or 0.5 % Triton-x100 (v/v) for either 10 or 30 min at 4 °C respectively. The cells were washed three times in PBS, blocked in 5 % BSA/PBS, for subsequent detection using SA-Alexa 633. Cells were visualised using a TCS SP laser scanning confocal microscopy (Leica Microsystems, Wetzlar, Germany) using a 60x objective. The He Ne laser (633 nm) was used and emission was collected at 645 +/− 20 nm using a standard PMT. Data were acquired in Leica Application Suite (Leica Microsystems).
Cellular subfractionation
NSC-34 cells were incubated with 20 μg/mL of aggregated SOD1 in PBS for 120 min at 37 °C. Post incubation the cytosolic (CEB), membrane (MEB), nuclear (NEB) and cytoskeletal proteins (PEB) were extracted from NSC-34 cells using a Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Fisher Scientific) according to the manufacturer’s instructions. Aliquots of cell extract (20 μg protein/lane) were separated under reducing conditions (5 % β-mercaptoethanol) using discontinuous TGX Stain-Free™ Precast Gels separating gels (BioRad). Proteins were then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) then blocked with heat denatured casein (HDC) in PBS (pH 7.4) for 1 h at 37 °C. To detect exogenously applied SOD1, sheep polyclonal anti-human SOD1 was used. To test the quality of the fractionation, rabbit anti-EEA1, anti- vimentin and mouse anti- actin antibody diluted in HDC/PBS for 1 h at 37 °C were used to probe the MEB and PEB fractions. Membranes were visualised using chemiluminescent substrate and Amersham Hyperfilm ECL (GE Healthcare, Little Chalfont, Bukinghamshire, UK). Images of films were collected using a GS-800 Calibrated Densitometer (Bio-Rad).
Membrane dye uptake
NSC-34 cells were treated with 20 μg/mL of soluble or aggregated wt or G93A SOD1, PBS alone, or a positive control containing 200 nM PMA in PBS for 2 h at 37 °C. The cells were then washed twice in PBS and incubated with 10 μM of FM® 1-43FX membrane stain in PBS for 7 min at 37 °C. Excess dye was removed by several washes in PBS and cells were returned to the incubator for 4 min. This procedure was repeated to give a total of 8 min of incubation in PBS. Post incubation, ice-cold PBS was added to stop endocytosis and prepare cells for fixation in 4 % (w/v) PFA/PBS (pH 7.4) for 20 min at 4 °C. Post fixation, cells were washed twice in PBS and incubated with 1x RedDot 2 for 10 min at RT.
Fluid phase uptake assays
Pinocytosis involves uptake of solutes from the extracellular medium. One well established solute is dextran. To quantify the amount of fluid phase solute uptake, NSC34 cells were treated with either 20 μg/mL of soluble and biotinylated aggregated wt and G93A SOD1, Httex146Q, α-synuclein, TDP-43, and α-lactalbumin in PBS alone or containing 200 nM PMA for 30 min at 37 °C. Prior to harvesting or fixation, cells were incubated for 15 min with 0.5 mg/ml 10 kDa 647-dextran (Invitrogen) at 37 °C. The cells were then placed on ice to stop dextran uptake and cells were washed three times with ice cold PBS and once with low pH buffer (0.1 M sodium acetate, 0.05 M NaCl, pH 5.5) for 10 min. The cells were then prepared for either flow cytometry or confocal laser scanning microscopy as described above. For flow cytometry, dextran uptake was displayed as fluorescence mean of three or more independent experiments.
Fixed cell antibody staining of iPSCs
The iPSCs were plated on matrigel-coated 8 mm coverslips at a density of 25,000 cells and cultured for 3 days before staining. Cells were fixed with 4 % (v/v) PFA at room temperature for 10 min, permeabilized with 0.5 % (v/v) TritionX-100 in phosphate buffered saline (PBS) and blocked with 5 % (w/v) bovine serum albumin (BSA) in PBS.
The iPSC colonies were stained with Oct3/4 (mouse 1:500) (Stem Cell Technologies) primary antibody overnight at 4C and anti-mouse Alexa Fluor 488 (1:1000) (Life Technologies) secondary antibody for 1 h at room temperature.
Fixed cell antibody staining of motor neurons
Cells were plated on coverslips coated with laminin (20 μg/mL) and fibronectin (10 μg/mL) at a density of 42,000 cells/cm2. Cells were fixed using 4 % (v/v) PFA at room temperature for 10 min. The cells were permeabilized with 0.5 % (v/v) Triton X-100 in PBS at room temperature for 15 min. Cells were blocked with 5 % (w/v) bovine serum albumin (BSA) in PBS at room temperature for 1 h. SMI32 primary antibody (Abcam) was diluted 1:800 in PBS 5 % BSA and incubated at 4 °C overnight. Secondary antibodies, Alexa Fluor 488 anti-sheep IgG antibody (1:1000 in PBS 5 % BSA) was incubated with the cells for 1 h at room temperature. Images of stained cells were taken on a Leica DMI6000B confocal microscope and acquired using the LAS AF 2.3.5 software.
Quantitative RT-PCR
RNA was extracted and purified from differentiated cell using the ISOLATE II RNA Mini Kit (Bioline, USA), as per manufacturer’s instructions. The purified RNA was quantified using a Nanodrop 2000C (Thermo Fisher Scientific, USA).
RNA was reverse transcribed into complementary DNA (cDNA) for subsequent analysis. Reagents for cDNA preparation were obtained from Promega (USA). Five μg of purified RNA was annealed to random primers (0.75 μg) and oligo dT primers (0.75 μg) by incubating at 65 °C for 4 min, followed by 1 min incubation on ice. For reverse transcription, Moloney-murine leukaemia virus reverse transcriptase (M-MLV RTase) (150 U), 96 nmol dNTPs, RNasin (60 U) and 1x MMLV RTase Buffer were added to the reaction mixture and then incubated at 37 °C for 100 min.
The primers for qRT-PCR were obtained from Sigma Aldrich (USA) (unless stated otherwise) and had the following sequences:
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acetylcholinesterase (AChE)
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forward: 5’-GGAACCGCTTCCTCCCCAAATTG-3’,
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reverse: 5’-TGCTGTAGTGGTCGAACTGGTTCTTC-3’; Homeobox 9 (MNX1)
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forward: 5’-GTTCAAGCTCAACAAGTACC-3’,
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reverse: 5’-GGTTCTGGAACCAAATCTTC-3’; GFAP
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forward: 5’-CTGGATCTGGAGAGGAAGATTGAGTCG-3’,
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reverse: 5’-CTCATACTGCGTGCGGATCTCTTTCA-3’; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward: 5’-GAGCACAAGAGGAAGAGAGAGACCC-3’, reverse: 5’-GTTGAGCACAGGGTACTTTATTGATGGTACATG-3’. The final qRT-PCR reaction consisted of 10 μL of SYBR Select Master Mix, 800 nM of each forward and reverse primer, 2 μL of cDNA in a final reaction volume of 20 μL. Each reaction was run in duplicate and a negative control (water) and no reverse transcription (RNA) control was included as well as a positive control using cDNA of human putamen. The amplification consisted of 40 cycles, of 95 °C for 15 s (activation step), 58 °C for 15 s (annealing step) and 72 °C for 1 min. A melting curve analysis was conducted to confirm the presence of the appropriate amplified target. The acquired data was normalized against quantitative expression levels of the housekeeping gene GAPDH and analyzed using the comparative threshold cycle method.
Preparation of giant unilamellar vesicles
The rapid evaporation method was used to prepare giant unilamellar vesicles for confocal microscopy as described in [36]. Briefly, soy L-α-phosphatidylcholine (Avanti Polar Lipids Inc) was dissolved in CHCl3:MeOH (2:1) to give a phospholipid concentration of 5 mM. Liposome buffer (50 mM HEPES, 107 mM NaCl, 1 mM EDTA, 0.1 M sucrose, pH 7.4, 2.5 mL) was then added to the lipid/solvent solution in a 50 mL round bottom flask and the two phases mixed by vigorous pipetting. The organic solvent was removed by rotary evaporator under reduced pressure (final pressure 44 mbar) for 5 min at 35 °C. The resulting liposome suspension was stored overnight at 4 °C prior to confocal microscopy studies.